There have not been successful and repeatable methods for in vitro embryo production in the dog. Up to date, only one blactocyst has been achieved on in vitro culture. Since reproductive physiology of the dog is different from that of other mammalian species, it seems that a suitable method for in vitro production of canine embryos is still far from being designed and routinely applied, and an effective protocol is needed. Therefore, the aim of the present study was to examine the effects of adding hormones sequentially, for mimicking the dog's in vivo endocrine milieu, on maturation of immature dog oocytes in vitro. At the end of the 96 h IVM period, nuclear maturation rates were evaluated by the aceto-orcein staining method. In comparison relating IVM rates, the sequential hormone addition was more beneficial on IVM rates (MI + MII) than the traditional hormone addition and control groups (48.1%, 38.9% and 23.0% respectively; P < 0.0001). As a result, hormone addition sequentially may be an effective approach for the IVM of the immature dog oocytes. We suggest that attempts to define the adequate conditions for IVM in the dog should extend towards this new perspective. (C) 2011 Elsevier Inc. All rights reserved.